Coating compositions for pathogen control in cotton

ABSTRACT

Coating compositions for cotton plant seed, wherein the coating composition comprises an organic carrier material and one or more biological agents that are active against one or more pathogens cotton plant.

The present invention relates to coating compositions including anorganic component and a biological agent for applying to cotton plantseed from which roots and shoots are capable of growing, uses of coatingcompositions on cotton plant seeds, methods of producing such coatingcompositions and seeds coated with such coating compositions. Inparticular, the invention relates to coating compositions that comprisean organic carrying material and biological agents selected fromchemicals and biological agents active against one of more plantpathogens selected from bacterial, fungal and arthropod pathogens thatinfest seeds of cotton plants.

Losses in yield in cotton crops are recorded annually and come about asa result of plant infestations due to pathogens such as bacteria, fungiand arthropods which can infest the plant at various stages ofdevelopment, such as at the seed stage. Agronomic losses due to pathogeninfestations remain high despite many defensive measures that have beendevised by man to combat such infestations. Such defensive measuresinclude the use of synthetic chemicals; the employment of geneticengineering of cotton plants; and the use of live biological agents thatare applied in the form of coatings, sprays and washes to seeds.

Pesticides in the form of chemical agents such as fungicides,bactericides and arthropodicides, typically in the form of insecticidesand/or acaricides may be applied to cotton crops in the form of soildrenches, seed treatments and the like. Such kinds of chemicaltreatments tend to be indiscriminate in their action and may adverselyaffect beneficial bacteria, fungi and arthropods as well as the plantpathogens at which such treatments are targeted.

When conventional pesticides are used as seed treatments the seeds arecoated with pesticide directly or the pesticide is applied to the seedin the presence of an inorganic carrier. Such seed treatments aretypically applied in liquid form or as wet slurry and subsequently theseeds are dried. Such treatments are mostly aimed at providing directprotection against pathogens such as arthropods and/or seed bornemicroorganisms and/or soil borne microorganisms that attack the seed.The high level of chemicals that are typically used introduces achemical load to the environment that may give rise to ecologicalconcerns.

One problem in applying a biological agent that is a chemical agent inconventional seed coating procedures is that the chemical agent istypically applied as slurry and this may give rise to an unevenapplication of the coating whereby the seeds are not fully coated or apercentage of the seeds, up to 20% depending on seed type and thecoating procedure employed, do not get fully coated. Furthermore, theseed coatings may not be uniform and this gives rise to physicalweaknesses in the seed coat and the coating may flake off.

A further problem arises when using biological agents that are selectedfrom beneficial live bacterial and fungal species that may be appliedconventionally to seeds, for example as spores in conjunction with aninorganic carrier in the form of particulate compositions or in the formof liquid compositions which may then be dried back, is that the appliedbiological agents rapidly lose viability. Without the intention of beingbound by theory, it is thought that as the seeds are dried off themicro-environment alters and the viability of applied live biologicalagents may be seen to decrease sharply and almost as soon as the appliedcomposition dries. The loss of viability of the biological agent istypically associated with the splitting of the fungal or bacterialspores which renders them non-viable.

It has now been found that by using an organic carrier material inconjunction with a biological agent, the viability of the biologicalagent is improved on cotton seeds, relative to the viability ofbiological agents applied to such seeds conventionally. Furthermore, thecoating of the seed is less susceptible to flaking off.

It is an object of the present invention to supply improved seedcoatings comprising biological agents for cotton plant seeds. This andother objects of the invention will become apparent from the followingdescription and examples.

According to the present invention there is provided a cotton plant seedcoating composition, wherein the said coating composition comprises atleast one organic carrier material in the form of particles wherein thecarrier material is selected from waxes having a melting point of ≧50°Centigrade and one or more biological agents that possess an activityagainst one or more pathogens of a cotton plant.

The cotton plant seed coating composition is applied to cotton plantseeds from which roots and shoots are capable of growing. For thepurposes of the present invention a cotton plant seed is one from whichroots and shoots are able to grow. Reference to “seed” and “seeds” isused interchangeably herein and means cotton seeds, typically viableseeds, to which compositions of the invention may be applied.

The organic carrier material is selected from organic materials that canbe applied to cotton plant seeds either as a powder wherein the powderparticles are of a pre-determined volume mean diameter, or in liquidform, such as an oleaginous formulation or as an aqueous formulation.

Generally, the composite particles of use in a dry powder composition ofthe invention possess a volume mean diameter of a certain size asdefined herein. To obtain particles of organic materials of a volumemean diameter applicable for use in the invention, organic materials inthe form of, for example, 1 to 5 kilogram blocks or tablets may bebroken up or kibbled into small millimetre-sized pieces (such as from 2mm-8 mm approximate diameter in size, for example from 4 mm to 6 mm) ina kibbling machine. The millimetre-sized pieces can then be passedthrough a comminuting means such as a standard mill, e.g. an ApexComminuting mill, and milled or comminuted into particles having anapproximate diameter in the range from 100 μm-500 μm, for example from250 μm-300 μm. The micron-sized comminuted particles can then be passedthrough a micronising apparatus, such as an AFG micronising air mill toobtain particles of a desired VMD range, such as from 15 μm-20 μm, thatis of use in the present invention. The skilled addressee willappreciate that such procedures for obtaining small particles are wellknown in the art. Preferably, dry powder compositions of the inventioncomprise composite particles having a volume mean diameter of ≧5 μm, forexample of 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm up to 40 μm or anyvalue thereinbetween. As stated herein, the volume mean diameter of thecomposite particles is typically ≧10 μm or ≧12 μm and may lie in therange from 10 μm to 200 μm and may have a value that lies anywhere thereinbetween, for example from ≧5 μm to 100 μm; or from ≧10 μm to 40 μm; orfrom ≧10 μm to 30 μm or any desired volume mean diameter value inbetween. Preferably, dry powder compositions of the invention compriseparticles having a volume mean diameter of ≧5 μm, for example of 8 μm, 9μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15μm and the like up to anyvolume mean diameter of choice, such as up to 200 μm or any volume meandiameter in between for example 40 μm or 30 μm. Such compositions areconsidered to be less of a thoracic hazard to humans and are not thoughtto be allergenic.

In liquid formulations, particles of a pre-determined volume meandiameter are suspended therein in a suspension formulation and appliedto the seeds which are then dried using conventional drying procedures.Preferably, the organic carrier material is applied to cotton plantseeds in a dry powder form, the particles of the organic carriermaterial may have a volume mean diameter of any conventional size asherein described. To such dry powders, chemicals of use againstarthropod pathogens such as insects, arachnids (e.g. mites) and ifappropriate, their larvae, eggs, or pupae; chemicals of use againstbacterial pathogens; and chemicals of use against fungal pathogens maybe added. Additionally, beneficial live biological agents may be addedto cotton plant seeds that are able to target bacterial pathogens of thecotton plant and/or to target fungal pathogens and/or arthropods of thecotton plant. Spores of choice of beneficial live biological agents suchas fungal conidia or hyphae or mycelia of fungi that do not form sporesor conidia-like structures may be added to dry powders of use in thepresent invention. Suitable organic carrier materials of use in theinvention are typically made up of organic materials such as waxeshaving a melting point of ≧50° C., more preferably of ≧60° C., and mostpreferably are made up of hard waxes having a melting point of ≧70° C.

Natural waxes of use in the present invention include carnauba wax,beeswax, Chinese wax, shellac wax, spermaceti wax, myricyl palmitate,cetyl palmitate, candelilla wax, castor wax, ouricury wax, wool wax,sugar cane wax, retamo wax, rice bran wax and the like.

Synthetic waxes of use in the present invention include suitable waxesselected from paraffin wax, microcrystalline wax, Polyethylene waxes,Fischer-Tropsch waxes, substituted amide waxes, polymerized a-olefinsand the like.

Mineral waxes of use in the invention include montan wax (e.g. Lumax®Bayer) ceresin wax, ozocerite, peat wax and the like.

Suitable organic carrier particles of use in the present invention maybe selected from waxes such as carnauba wax, beeswax, montan wax,Chinese wax, shellac wax, spermaceti wax, myricyl palmitate, cetylpalmitate, candelilla wax, castor wax, ouricury wax, wool wax, sugarcane wax, retamo wax, and rice bran wax. Such waxes typically display ahigh enthalpy of lattice energy during melt. Preferably the organiccarrier material is carnauba wax which may be applied in liquid form,typically in the form of a suspension, or in powder form as discreteparticles. Generally, the particles of use in the invention possess avolume mean diameter as herein described.

Additionally, the organic carrier particles of use in compositions ofthe invention may contain other components such as additives selectedfrom UV blockers such as beta-carotene or p-amino benzoic acid,colouring agents such as optical brighteners and commercially availablecolouring agents such as food colouring agents, plasticisers such asglycerine or soy oil, antimicrobials such as potassium sorbate,nitrates, nitrites, propylene oxide and the like, antioxidants such asvitamin E, butylated hydroxyl anisole (BHA), butylated hydroxytoluene(BHT), and other antioxidants that may be present, or mixtures thereof.The skilled addressee will appreciate that the selection of suchcommonly included additives will be made depending on end purpose, andperceived need.

Liquid formulations of the invention may be formulated as an aqueousformulation or as an oleaginous formulation, depending on design.Aqueous formulations may include surfactants selected from commerciallyavailable surfactants such as Libsorb, Silwet L77, Tween 80, Torpedo II,Newmans T80, Fortune, Guard, Rhino, Biopower, and the like.

Oleaginous formulations, that is to say oil-based formulations, maycontain any oil suitable for use in the present invention which may beselected from petroleum oils, such as paraffin oil, and vegetable oilssuch as rapeseed oil, soybean oil, sunflower oil, palm oil and the like.Oil formulations of use in the invention contain organic carrierparticles as described herein and these in turn may be admixed with flowagents such as hydrophilic precipitated silicas, for example Sipernat383 DS, Sipernat 320, EXP 4350, and Sipernat D-17 and the like. Suchfree-flowing agents may be dispersed in oils, for example, foranti-foaming purposes.

The skilled addressee will appreciate that where an aqueous or an oilformulation may be used to apply biological agents of use in theinvention, the liquid element should be removed from the coated seedafter coating is achieved, for example by drying off using conventionaldrying processes.

A biological agent for the purposes of the present invention is one thatcan be used to control the population of a plant pathogen of a cottonplant, and may be selected from chemical fungicides, arthropodicidessuch as insecticides and acaricides, bactericides; or a mixture of twoor more thereof; and/or from live biological agents that are able tocontrol the population of one or more seed or soil borne pathogens of acotton plant seed. Preferably, the population of the soil borne pathogenon or in the immediate vicinity of the cotton plant seed is reducedeither by the biological agent rendering it unable to reproduce and/orby killing it. Examples of biological agents of use in the presentinvention include chemicals for use on cotton plant seeds selected fromarthropodicides, such as insecticides and acaricides, fungicides andbactericides commonly employed in the art. Suitable examples of suchchemicals include nicotinoid insecticides such as imidacloprid[(E)-1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine],methylcarbamate insecticides such as methiocarb [4-methylthio-3,5-xylylmethylcarbamate], oxime carbamate insecticides such as thiodicarb[(3EZ,12EZ)-3,7,9,13-tetramethyl-5,11-dioxa-2,8,14-trithia-4,7,9,12-tetraazapentadeca-3,12-diene-6,10-dione],and thiazole insecticides such as clothianicidin[(E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine],Thiamethoxam(EZ)-3-(2-chloro-1,3-thiazol-5-ylmethyl)-5-methyl-1,3,5-oxadiazinan-4-ylidene(nitro)amine;suitable fungicides for use on seeds in accordance with the inventioninclude those selected from acyl amino acid fungicides such as mefenoxam[methyl N-(methoxyacetyl)-N-(2,6-xylyl)-D-alaninate], pyrrole fungicidessuch as fludioxinil[4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1H-pyrrole-3-carbonitrile],thiazole fungicides such as thiabendazole [2-(thiazol-4-yl)benzimidazoleor 2-(1,3-thiazol-4-yl)benzimidazole], and conazole fungicides such asdifenoconazole[3-chloro-4-[(2RS,4RS;2RS,4SR)-4-methyl-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-2-yl]phenyl4-chlorophenyl ether].

The skilled addressee will appreciate that compositions of the inventionmay also be added direct to the soil or growing medium into which cottonseeds are to be planted. Such compositions may be added as powders andmixed with the soil or applied as liquid suspensions using conventionalprocedures.

Soil borne pathogens for the purposes of the present invention are onesthat are able to colonise the seed cuticle and/or ones that populate thesoil and which are capable of acting on cotton seeds. Such soil bornepathogens are typically bacteria and/or fungi. Examples of soil bornebacterial and fungal pathogens that attack cotton plants includeAgrobacterium tumefaciens, Xanthomonas campestris pv malvacearum,Erwinia herbicola, Rhizoctonia spp. e.g. R. solani, Pythium spp,Sclerotium spp. such as S. rolfsii, Fusarium spp. such as F. oxysporumf. sp. vasinfectum, Phytophthora spp., Verticillium spp. such as V.dahliae, Phoma spp. such P. exigua, Alternaria spp. such as A.macrospora, A. alternata, and the like.

According to a further aspect of the invention there is provided use ofan organic carrier wherein the organic carrier is made up of particlesof wax in the manufacture of a cotton seed coating composition thatincludes a biological agent as defined herein above. The organic carrierparticles are selected from natural waxes, synthetic waxes, and mineralwaxes having a melting point of ≧50° C., more preferably of ≧60° C., andmost preferably are made up of hard waxes having a melting point of ≧70°C. Waxes of use in the invention may be selected from carnauba wax,beeswax, montan wax, Chinese wax, shellac wax, spermaceti wax, myricylpalmitate, cetyl palmitate, candelilla wax, castor wax, ouricury wax,wool wax, sugar cane wax, retamo wax, and rice bran wax or a mixture oftwo or more thereof, and preferably, the seed coating that is usedincludes particles of carnauba wax. Preferably, in this aspect of theinvention, the organic carrier particles have a mean volume diameter ≧5μm as herein described.

In a further aspect of the invention there is provided use of wax as anorganic carrier in dry particulate form in a cotton seed coatingcomposition of the invention as herein described. Suitable organiccarrier particles employed in this aspect of the invention are selectedfrom natural waxes, synthetic waxes, and mineral waxes having a meltingpoint of ≧50° C., more preferably of ≧60° C., and most preferably aremade up of hard waxes having a melting point of ≧70° C. Suitable organiccarrier particles may be selected from carnauba wax, beeswax, montanwax, Chinese wax, shellac wax, spermaceti wax, myricyl palmitate, cetylpalmitate, candelilla wax, castor wax, ouricury wax, wool wax, sugarcane wax, retamo wax, and rice bran wax or a mixture of two or morethereof. Preferably, the seed coating composition comprises organiccarrier particles that are of carnauba wax. Organic carrier particlesemployed in this aspect of the invention have a mean volume diameter ≧5μm as herein described, such as in the range of ≧10 μm to 200 μm.

In a still further aspect of the invention there is provided a method ofmanufacturing a cotton seed coating composition as herein described thatcomprises

-   -   1) selecting an organic carrier material wherein the carrier        material is selected from waxes having a melting point of ≧50°        Centigrade;    -   2) comminuting said organic carrier material into particles of a        desired volume mean diameter ≧5 μm, such as in the range from        ≧10 μm to 200 μm; and    -   3) adding biological agent to the product particles of step 2).

The biological agent of use in this aspect of the invention is typicallyselected from a chemical agent which is an insecticide or an acaricideor a mixture thereof, or a chemical fungicide or a fungus species and/ora bacterium species or a mixture of two or more thereof.

Examples of live biological agents (also known as biocontrol organismsor biocontrol agents) that are commonly referred to in the art as“biological antagonists” that may be used in seed coating compositionsof the present invention include Pseudomonas spp. such as P.fluorescens, Trichoderma spp. such as T. viride (in seed dressings isactive against Rhizoctonia solani, Macrophomina phaseolina and Fusariumspp.) and T. harzanium e.g. Trichoderma harzianium Rifai strain KRL-AG2(T-22)(available from Bioworks Inc, Geneva, USA), Burkholderia cepaciatype Wisconsin (available from Stine Microbial Products, Memphis, USA)Burkholderia cepacia (available from Soil Technologies Corp., Fairfield,USA), Bacillus spp. such as B. subtilis e.g. B. subtilis GB03 (availablefrom Gustafson Inc., Plano, USA), Agrobacterium radiobacter Strain 84(available from AgbioChem Inc, Florida, USA), and the like.

Suitable fungicides that may be used in seed treatments on cotton seedsinclude those fungicides selected from acyl amino acid fungicides suchas mefenoxam [methyl N-(methoxy acetyl)-N-(2,6-xylyl)-D-alaninate],pyrrole fungicides such as fludioxinil[4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1H-pyrrole-3-carbonitrile],thiazole fungicides such as thiabendazole [2-(thiazol-4-yl)benzimidazoleor 2-(1,3-thiazol-4-yl)benzimidazole], conazole fungicides such asdifenoconazole[3-chloro-4-[(2RS,4RS;2RS,4SR)-4-methyl-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-2-yl]phenyl4-chlorophenyl ether] and the like.

Suitable examples of such chemicals further include nicotinoidinsecticides such as imidacloprid[(E)-1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine],methylcarbamate insecticides such as methiocarb [4-methylthio-3,5-xylylmethylcarbamate], oxime carbamate insecticides such as thiodicarb[(3EZ,12EZ)-3,7,9,13-tetramethyl-5,11-dioxa-2,8,14-trithia-4,7,9,12-tetraazapentadeca-3,12-diene-6,10-dione],thiazole insecticides such as clothianicidin[(E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine], andThiamethoxam (EZ)-3-(2-chloro-1,3-thiazol-5-ylmethyl)-5-methyl-1,3,5-oxadiazinan-4-ylidene(nitro)amine and the like.

The organic carrier material in this aspect of the invention may beselected from carnauba wax, beeswax, montan wax, Chinese wax, shellacwax, spermaceti wax, myricyl palmitate, cetyl palmitate, candelilla wax,castor wax, ouricury wax, wool wax, sugar cane wax, retamo wax, and ricebran wax or a mixture of two or more thereof. Preferably, the selectedcarrier material is carnauba wax.

In a further aspect of the invention, there is provided a cotton seedcoating composition, such as a seed coating composition produced by themethods as described herein.

In a further aspect of the invention there is provided a cotton seedcomprising a coating composition as described herein..

In a further aspect of the invention there is provided a method ofcoating cotton seed with a coating composition that comprises an organiccarrier material and a biological agent that has an activity against acotton plant pathogen selected from a fungal pathogen, a bacterialpathogen and an arthropod pathogen so as to limit damage by the saidpathogen to cotton plants, the method comprising adding the saidbiological agent to an organic carrier material wherein the organiccarrier material is in dry particulate form, mixing the two componentstogether and applying the resulting composition to cotton seeds.

The treatment composition is applied to the plant seed in dryparticulate form or liquid form as hereinbefore described, andpreferably in dry particulate form. The organic carrier material in thisaspect of the invention is selected from waxes as herein described, suchas carnauba wax, beeswax, montan wax, Chinese wax, shellac wax,spermaceti wax, candelilla wax, castor wax, ouricury wax, and rice branwax or mixtures of two or more thereof. Preferably, the organic carriermaterial is carnauba wax.

The treatment composition in this aspect of the invention typicallyincludes one or more biological agents selected from insecticides andacaricides, fungicides, bactericides and live biological agents asherein before described.

There now follow examples and that illustrate the invention. It is to beunderstood that the

EXAMPLES SECTION

Control of Fusarium oxysporum spp. [United Kingdom National CultureCollection (UKNCC)] on cotton (Gossypium hirsutum) by means of seedtreatments using examples of the antagonists: Trichoderma harzianum,Pseudomonas fluorescens and Bacillus subtilis [United Kingdom NationalCulture Collection (UKNCC)].

Fusarium Wilt

Symptoms

Symptoms of Fusarium wilt include a general wilt, which is especiallyevident in a dry environment at temperatures above 20° C., and yellowingand necrosis of lower leaf margins. The vascular system of infectedplants is brownish to black. This is most apparent in the lower stem andupper taproot. The discoloration is generally continuous in contrast tothe speckling nature of the discoloration in plants affected byVerticillium wilt.

In seedlings and young plants, cotyledons and leaves wilt and drop,resulting in bare stems. In severe cases, plants die. In mildly affectedplants, lower leaves develop symptoms but plants survive, but withreduced vigour. Certain strains of the causal fungus only cause symptomswhen plants are also infected with the root knot nematode. In thosecases, galls are usually prevalent on lateral roots.

The fungus sustains itself on the outer surface of roots of many cropsand weeds and survives indefinitely in soil. In addition, the pathogenis seed-borne in cotton, which accounts for long-distance spread, and isalso spread whenever infested soil is transported on boots, farmequipment, in flood irrigation, etc. However, seed of cotton is oftentreated with chemicals to remove the lint that reduce micro-organisms onthe seed surface.

Disadvantages of Conventional Seed Treatment

-   -   i) Limited dose capacity—The amount of pesticide that can be        applied is limited by how much will actually stick to the seed.    -   ii) Limited duration of protection—The duration is often short        due to the relatively small amount of biological agent (e.g.        chemical) applied to the seed, dilution of the biological agent        as the plant grows, and breakdown of the biological agent.    -   iii) Limited shelf life of treated seed—Producing excess treated        seed is undesirable because the shelf life of treated seed may        be limited. Surplus treated seed cannot be sold for grain. This        is a particularly serious limitation for seeds such as cotton,        where seed germination and vigour decline relatively quickly.

All three of these limitations may be overcome or significantly reducedthrough the inclusion of carnauba wax particles as a carrier for abiological agent, in this case dormant microorganisms that are appliedto seeds. Under favourable conditions, the microorganisms grow andcolonize the exterior of the developing seed or seedling. Biologicalagents may help in reducing seed decay, seedling diseases, or root rot.

The following tests are performed to examine the potential effect of theinclusion of carnauba wax particles.

Phase One—Isolate Cultures

1. Culture Maintenance

Records are kept with each isolate sub-culture being assigned anaccession number. All plates and slides relating to that sub-culture arelabelled with an accession number.

In addition, permanent lactophenol (LP) mount slide are made from eachof the original cultures and file for reference purposes.

No more than three generations of sub-culture occur before passagingthrough a living host and re-isolating in order to maintain the fitnessof the organism.

Sub-cultures are stored for future use on Potato Dextrose Agar (PDA) at4° c.

Each isolate is assigned an accession number and sub-cultures arelabelled with that number.

DNA is extracted for identity verifications and stored at −20° C. Areference sample of the pure culture is stored on glycerol at −20° C.Upon completion of the experiment DNA identification of the culture isrepeated to confirm that the organism has not mutated during the courseof the work.

2. Culturing of the causal agent

Isolation of a pathogenic fungus from diseased tissue into pure cultureis one of the standard techniques in identifying and describing adisease. It is an essential step in proving the pathogenicity ofpreviously un-encountered organisms.

Techniques commonly involve:

-   -   a. Surface-sterilisation treatment    -   b. Plating (possibly on selective medium) of samples of diseased        tissue, with appropriate precautions.    -   c. Sub-culturing to get pure cultures.

3. Purification of Cultures

Small disinfected root pieces of an artificially inoculated plant arecultured on water agar. The fungal colonies that appear most frequentlyare likely the target pathogen. Several saprophytes may also be presentin infected plant tissues and they may grow into the medium with theprincipal pathogen. Routine surface-sterilisation consists of wiping thetissue with (or immersing in) 0.1% solution of sodium hypochlorite(NaOCl—also referred to as “NaClO”) followed by rinsing with steriledistilled water. To obtain a pure culture of the pathogen, a smallsample is taken from the growing edge of a colony with a flamed loop orscalpel and streaked over the surface of a pre-poured plate of PDA. Theinclusion of chloramphenicol (a bacteriostatic anti-microbial) at 30mg/l reduces the risk of bacterial contamination. As the streakprogresses over the agar, fungal spores are separated until singlespores are obtained from which separate colonies will grow.

Repeat this procedure until pure cultures are obtained.

4. Single Spore Isolation

Single spore isolations are important to investigate pathogenicvariability. An inoculum of spores is placed in a tube containing 10 mlof sterile water. This spore suspension is streaked along a marked lineon the surface of a thin tap water agar medium, and incubated at 22° C.After 24hr incubation, select germinated spores using a stereoscopicmicroscope and transferred one spore at a time to another agar plate.

5. Slide Preparation for Microscopic examination and reference

Identification of the pathogen, rather than the disease, requiresmicroscopic examination of infected tissue. The tissue may be sectionedor surface scraped and then mounted in water/lactophenol. Fungalstructures seen macroscopically may be separated from the host tissue tobe examined and identified. Identification depends on spore formationand therefore infected material will be incubated in a moist environmentovernight prior to examination in order to encourage sporulation. Cottonblue stain is added to the lactophenol in order to highlight fungalstructure. The specimen is placed in a drop of satin on a glass slideand gently warmed by passing through a low flame for a few secondsbefore mounting in lactophenol.

Whole mount sections can be cleared and stained for ease ofidentification using the following method:

Clear leaf disks are obtained by heating them in a tube in lactophenoluntil clear (up to 20 minutes), without boiling. Stain by heating in0.5% cotton blue in lactophenol on a slide for 5-10 minutes. Rinsethoroughly in lactophenol and mount in the same.

6. Growth and Media

Sub-cultures are assessed for growth and germination at a range oftemperatures, 15° C., 22° C. and 29° C. A range of media is examined forsuitability. Whilst PDA is generally suitable for most fungal species ithas been found that use of a low nutrient agar, such as tap-water agar,reduce prolific growth and can encourage sporulation. Therefore PDA,tap-water agar, and a selective media from literature, Czapek's Dox agar(Dawson (1962) Saboutaudia 1. 214-219), are included within theassessment trials.

A 5 mm diameter disk is cut from the margin of an actively growingculture using a flamed cork borer. This is placed upside down in thecentre of the pre-poured media plates. Five replicates are made for eachmedia type and temperature (45 plates in total). Complete randomisationis applied to plates in each incubator. Plates are observed until oneculture succeeds in completely covering the plate in any one media. Atthis point the following measurements are taken: fungus colony diameter,colour and margin. In addition, the level of sporulation is recorded.

Five 5 mm disks are cut from each plate using a flamed cork borer andsuspended in 20 ml of distilled water (+0.05% Tween 20®). The sample isthen sonicated for 2 minutes to release the spores and then vortexed toaid the formation of a uniform spore suspension. Samples are assessedfor spore concentration using an Improved Neubauer haemocytometer usingstandard counting methodology.

The mean for each media type is calculated and ANOVA is applied toexamine the results for significant differences.

Phase Two—In vitro studies:

1. Screen microorganisms and carnauba wax to determine interactions

In order to explain effects observed the microorganisms, pathogens andantagonists, will be screened against carnauba wax to identify anycarrier only effect. This will enable the determination of treatmenteffect as well as any synergy occurring as a result of the use of usingan antagonist with carnauba wax particles.

-   -   a. Plates of appropriate media are used based on the findings of        the experiment above.

Air-milled carnauba wax is sterilised using the autoclave and thenground using a twin blade mill, producing particles with an approximateVMD of 130 μm. The sterilised media is then cooled to 50° C. (moltenstage). The carnauba wax is then incorporated into the media. Twoconcentrations of carnauba wax are tested; 1 g/l and 10 g/l. A 5 mmdiameter disk is cut from the margin of an actively growing cultureusing a flamed cork borer. This is placed upside down in the centre ofthe pre-poured media/carnauba wax plates. Five replicates are made foreach concentration and incubated at the optimum temperature forgrowth/sporulation (as determined in previous experiment). Growth ratesand characteristics are compared to the controls using data from theGrowth and Media experiment above.

Differences are analysed using ANOVA.

-   -   b. Disks of the pathogen and antagonists are dusted with        different carnauba wax treatments and put on appropriate media.        The carnauba wax particles need to be free of microorganisms to        be able to carry out this experiment. Growth of treated and        untreated organisms are compared.

2. Investigate antagonist action against pathogens

i. Effect of antagonists on viability of F. oxysporum mycelium (in vitroassay I) All antagonistic isolates are tested in a dual culture assayagainst pathogenic fungi on PDA or alternative pre-defined media. Agarplugs of F. oxysporum and the antagonist isolate to be tested arearranged 7 cm apart on 9 cm agar plates. Inhibition zones and zones ofoverlapping are assessed after 7 days incubation at 19° C., 25° C. and31° C. Where an antagonist overgrows the mycelium of F. oxysporum, thezone of hyphal interaction between both is investigated microscopically(100×). Fungal strains without a microscopically visible effect onmycelium of F. oxysporum are excluded from further experiments.Furthermore, the viability of F. oxysporum in the region of interactionis tested by transfer of mycelial discs onto water agar plates 5 daysafter first contact. The F. oxysporum mycelium is assessed as viablewhen the growth of typical hyphae is observed microscopically (100×).Each experiment is repeated three times with three samples perreplicate.

ii. Effect of antagonists on germination of F. oxysporum sclerotiaproduced in vitro (in vitro assay II)

Sclerotia of F. oxysporum of uniform size are placed on a 6 day oldculture (PDA, 20° C.) of the fungal antagonist. After incubation for 14,28 and 35 days at 20° C., eight sclerotia per replicate (threereplicates per antagonist) are transferred from the agar plate ontowater agar. Mycelial growth from these sclerotia will be assessed undera light microscope (100×).

3. Confirmation of pathogenicity

Steps to perform Koch's postulates (Koch 1890, criteria designed toestablish a causal relationship between a causative microbe and adisease)

-   -   a) Describe the symptoms expressed by the diseased crop plants.    -   b) Isolate the suspected pathogen—the same cultures should be        isolated from plants with similar symptoms    -   c) Obtain a pure culture and use it to inoculate healthy plant        material.    -   d) Observe the symptoms expressed by the inoculated        plants—symptoms should be the same as those observed originally        in the crop plants.    -   e) Re-isolate the pathogen from the newly diseased material. The        culture should be the same as the original purified culture.

i. Indirect Application—Plant

-   -   Using healthy plants—soil can be inoculated directly using a        spore suspension made from a pure agar culture or from a culture        grown in flasks. A fungal spore or bacterial suspension can be        added post-emergence so that the root system is drenched by the        suspension. Plants are then observed over 7 days and symptoms        recorded. Koch's Postulates are applied in order to confirm that        the symptoms relate to the inoculated pathogen.

ii. Direct Application—Seed

-   -   Inoculum for preparing spore suspensions is grown on water agar        containing sterile seeds. Fungal spores and hyphae or bacterial        spore and vegetative growth are scraped from the colony and        transfer to sterile water. This spore suspension is then applied        to seeds and mixed to ensure a uniform distribution. Seeds are        then:        -   Placed on moist filter paper and incubated at optimum growth            temperature for 5 days.        -   sown in heat sterilised potting compost and incubated in a            propagator at optimum growth temperature for 7 days

Symptom expression and germination is recorded for both sets ofexperiments and Koch's postulates applied

4. Carnauba Wax/Antagonist co-location analysis

A dry powder formulation of spores is produced using a spore separator.Moisture content of the formulation is reduced to below 5% using adehumidifier and silica beads. Spore concentration is determined using aNeubauer haemocytometer and standardised counting methodology.

Steps in Air Milling in Boyes Micronisation Process (for carnauba waxparticles with a VMD of approx. 15 μm and 75 μm, respectively)

1. 2 kg carnauba wax blocks are first kibbled into approximately 4 to 6mm pieces in a KT Handling Ltd Model 04 kibbler (serial no. 729/C)following the manufacturer's instructions.

2. The kibbled pieces are then passed through an Apex Construction LtdModel 314.2 Comminuting Mill (serial no. A21306) and reduced further insize to a range of 250 to 300 um.

3. The comminuted particles are then passed through a Hosokawa MicronLtd Alpine 100AFG jet mill (serial no. 168092) following themanufacturer's instructions, setting the mill at a suitable speed (aspeed of 8000 rpm for particles having a VMD of 15 μm or at a speed of2500 rpm for particles having a VMD of 75 μm), with a positive systempressure of 0.03 bar.

4. The grinding air is to be kept to 6 bar, the system rinsing air flowand Classifying Wheel gap rinsing air are both to be set at a minimum of0.5 bar and no more than 0.75 bar, the cleaning air filter is toregister a delta of no more than 5 bar to achieve a final particle sizewith a VMD of 15 um or 75 μm as required.

Entostat was combined with oilseed at three loadings (see below).

Two sizes of carnauba wax particle having VMDs of 15 μm and 75 μm,respectively are examined in combination with the spore formulation attwo different ratios (1:3, 2:2). Samples of the carnauba wax/sporemixture are analysed using electron photomicroscopy to determine theco-location effect. Any variation observed is recorded.

In addition, both sizes of carnauba wax referred to, are mixed with ahomogenised sample of mycelium and examined as described above.

5. Carnauba Wax Particle loading

Carnauba wax particle adhesion to seeds is approximated through the useof photomicroscopy (qualitative) and fluorometric analysis(quantitative). Two sizes of carnauba wax particles (with 1% glo-brite)are used having a VMD of 15 μm and 75 μm, respectively.

Four combinations: Two ratios of carnauba wax/spore formulation,together with one mycelial and a vehicle control (carnauba wax only),makes a total of eight treatments. Treatments are applied to 10 g ofseed and replicated three times. Three subsamples are taken from eachreplicate and the mean used in analysis.

For fluorometric analysis three 1 g samples are each be added to 5 ml ofethanol and sonicated to aid the release of the carnauba wax particlesfrom the seeds. Samples are analysed using a Perkin Elmer L55Fluorometer (Perkin Elmer, Mass., USA). Statistical analysis ofvariation between treatments is performed using ANOVA.

Seed size and architecture varies greatly between crop species and thisinfluences application rates and method. A homogeneous mix is attainedthrough tumbling seed and carnauba wax formulation in a cylinder,adapted to produce lateral mixing/tumbling through the inclusion ofangled interior vanes, placed on a Wheaton roller for 5 minutes.

Phase Three—In vivo:

F. oxysporum, together with the most successful antagonist model is usedin a series of in vivo experiments. The basic design is a split-plotexperiment with temperature being the main plot factor (19° C., 25° C.and 31 ° C.) and carnauba wax/antagonist ratio (3 treatments: 2× spore,1× mycelial) being the sub-plot. Four homogeneous mixes of eachtreatment are prepared using the method described above and theserepresent the replicates.

Treatments:

-   -   1) Application rate 1—7.5×10⁶ conidia kg⁻¹    -   2) Application rate 2—7.5×10⁸ conidia kg⁻¹    -   3) Application 3—Mycelia    -   4) Control 1—Vehicle control (Carnauba wax only)    -   5) Control 2—no treatment

Mixes (true replicates): A, B, C, D

Subsamples of each mix: α, β, γ

Mixes and treatments are arranged according to a Randomised CompleteBlock design.

Pot studies

Each temperature (growth chamber) contains 60 plant pots.

Treated seed is sown in accordance with supplier's recommendation.Soil/compost (1:1 John Innes No.2 and Potting compost) is heatsterilised prior to inoculation with 10 ml of F. oxysporum sporesuspension and thoroughly mixed before sowing.

Plants are placed in the growth chambers for a period of 21 days withobservations of symptom expression made every 48 hours post emergence.Water is applied through capillary matting twice daily.

After 21 days plants are removed from their pots and the followingassessment measurements taken:

-   -   % germination    -   % pre-emergence damping off    -   % post-emergence damping off    -   Root weight    -   Shoot weight

In addition, symptom expression is assessed based on a damage scale.

Means of the measurements taken from the subsamples α, β, γ are comparedfor each treatment using ANOVA.

Samples are taken from 5 plants exhibiting symptoms and Koch'sPostulates applied to confirm the causal organism (by comparison to thereference slide of the master culture). The experiment will be repeated.

Second Example

Relating to:

Control of Rhizoctonia solani (United Kingdom National CultureCollection (UKNCC) on cotton (Gossypium hirsutum) by means of seedtreatments using fludioxonil.

Experimental Design: as for the Pot Study in Example 1, above.

Carnauba wax is melted using copper pans. During cooling fludinoxonil isadded at 1% of the mass of the carnauba. This mixture is allowed tosolidify before chipping and processing through a mill (as describedabove set at a milling speed of 6000 rpm), to produce particles with aVMD of 25 μm.

Treatments for the Pot Study—

Control 1—Vehicle control (Carnauba wax only)

Control 2—no treatment

Treatment 1—1% fludinoxonil carnauba wax at 10 g per kg of seed

Treatment 2—1% fludinoxonil carnauba wax at 3.2g per kg of seed

Assessment and analysis as with previous Pot Study

Third Example

Control of Limonius spp. (Coleoptera: Elateridae) Also known aswireworms, the larval form of the click beetle) on cotton (Gossypiumhirsutum) by means of seed treatments using thiamethoxam.

Early-season wireworm damage consists of hollowed- out seeds wherelarvae have entered during germination. Seedling plants also can beinjured or killed by larvae tunneling into the plant below the soilline. Occasionally, wireworms bore into the stalks of larger plants andtunnel in a few inches, but the damage is not significant.

Experimental Design—as Pot Study above

Carnauba wax is melted using copper pans. During cooling thiamethoxam isadded at 1% of the mass of the carnauba. This mixture is allowed tosolidify before chipping and processing through a mill to produceparticles with a VMD of 25 μm.

Treatments for the Pot Study—

Control 1—Vehicle control (Carnauba wax only)

Control 2—no treatment

Treatment 1—1% thiamethoxam carnauba wax at 4.2 g per kg of seed

Treatment 2—1% thiamethoxam carnauba wax at 1.3 g per kg of seed

Empty pots are lined with a nylon mesh screening material before fillingwith potting soil. A wire frame is constructed and the nylon meshed tiedoff over the frame to provide a caged experimental arena designed sothat the insect cannot escape the treated area.

Seeds are allowed to germinate for three days before adding five 3^(rd)instar larvae to the soil surface of each pot before resealing the meshcage.

Observations are made over 21 days.

Plants are assessed for:

-   -   % germination    -   Damage    -   Root weight    -   Shoot weight

Suppression of causal agents of fungal disease in Cotton (Gossypiumhirsutum) using a seed coating comprised of Trichoderma sp. and carnaubawax particles

The potential for Trichoderma sp. (Ascomycota) as a biocontrol agent inthe defence against plant pathogens is known.

Trichoderma hyphae are capable of penetrating the hyphae of other fungiand extracting nutrients from within, resulting in the suppression andeventual death of the host. Trichoderma exhibits rapid mycelial growthand is capable of out-competing other fungi for nutrients.

There are several commercially available formulations of Trichodermamarketed as crop protection products. These are commonly supplied as awettable powder formulation and applied to the area of cultivation as adrench. The disadvantage of this form of application is that it isnecessary to treat the entire cultivation area, whereas it is the regionimmediately surrounding the seed or plant that requires the treatment.The larger the number of conidia delivered to this area the greater thelevel of control they are able to impart. Therefore a targetedapplication system able to deliver sufficient conidia to the requiredarea offers a distinct advantage in the use of Trichoderma overconventional applications.

Experimental Aim: To assess the potential use of Entostat as aseed-coating technology for the delivery of beneficial microbes

Methods

Trichoderma harzianum was tested alongside Entostat on cotton. The studywas not replicated therefore statistical analysis has not been applied.

Haemocytometer counts, method as described below, were used to assessthe spore quantity in the wash from treated seed using 0.05% Tween²⁰solution.

Spore density of Trichoderma was 10⁹ spores per gram.

Thousand seed weight of cotton used was 113.49 g.

Treatments

The following treatments were used (per 100 seeds):

-   -   1. Trichoderma only (0.016 g)    -   2. Trichoderma (0.016 g)+Entostat (0.034 g)    -   3. Trichoderma (0.034 g)+Entostat (0.066 g)    -   4. Trichoderma (0.034 g)+Entostat (0.034 g)    -   5. Trichoderma only (0.034 g)    -   6. Entostat only (0.034 g)    -   7. Entostat only (0.066 g)

Spore Count Treatment per seed Trichoderma: 0.016 g 2.7 × 10⁵Trichoderma: 0.016 g + Entostat: 0.034 g 2.7 × 10⁵ Trichoderma: 0.034g + Entostat: 0.034 g 4.2 × 10⁵ Trichoderma: 0.034 g + Entostat: 0.066 g6.3 × 10⁵ Trichoderma: 0.034 g 4.5 × 10⁵

NB. Clumping of spores was observed in all treatments and this couldaffect the counting accuracy, with a tendency towards underestimation.It is notable that the inclusion of Entostat at the higher rate (0.066g) results in 50% increase in the spore count compared to the samenumber of Trichoderma spores combined with Entostat at 0.034 g.

1. Baseline data: seed coating techniques

-   -   1.1. Seed Coating. Trichoderma harzianum (containing 7.75×10⁹        colony forming units g⁻¹ Sylvan Bio, Loches, France) with a        germination percentage of 95% was applied to cotton supplied by        Herbiseed, (Twyford, UK) using carnauba wax particles with a VMD        of 9.7 μm (obtained using the process described above with a        mill speed set at 12,500 rpm). A target loading was set at 10⁵        conidia per seed based on information obtained from literature.

Carnauba particles were mixed with the dry conidia powder at differentratios and applied 0.01 g (0.2% by mass) directly to dry seed, 5 g ofseeds per concentration. For each concentration, four batches of 10seeds were used for evaluation of conidia loading.

Conidia to carnauba ratios used were:

100% Conidia, 50% Conidia, 25% Conidia and 9% Conidia with the remainderin each case being made up of carnauba wax particles.

1.2. Enumeration. Direct enumeration to determine conidia loading ofseeds was done through the use of a haemocytometer (Improved Neubauer,Hawksley, Lancing, UK).

Inoculum: Preparation of suspension.

Propagules are usually formulated in a water carrier, although thosewith hydrophobic cell walls (such as Trichoderma) are not readilysuspended in water. To uniformly suspend hydrophobic propagules in waterit is necessary to sonicate and/or use mechanical suspension methods.Mechanical suspension of propagules using micropestles provides goodsuspension of conidia in water without causing damage to cells. Asurfactant may also facilitate suspension of propagules (Tween20 at0.05%). To suspend hydrophobic conidia, harvested conidia are placed ina 1.5 ml microcentrifuge tube, ≈0.5 ml of sterile water is added to thetube, the micropestle is inserted into the tube , and the conidial massis gently agitated with the micropestle by hand. The micropestle is thenattached to the motor (e.g. Kontes, Argos pellet pestle motor) and thesuspension is vigorously agitated while moving the pestle in and up anddown, and side to side motion, circa. 30 seconds. Since thehaemocytometer method does not distinguish between viable and non-viablepropagules, it is necessary to determine spore viability so that dosescan be prepared on the basis of viable propagules.

Seed washes and enumeration of Trichoderma loadings were done on 4batches of seeds per treatment. Inoculum was washed from seeds byplacing into 1 ml sterile 0.05% Tween²⁰ (or substitute—similar non-ionicsurfactant/dispersal agent) in a Eppendorf tube and vortexing for 30seconds to remove conidia from the seed surface. Samples were thensonicated for two minutes to break up any conidial clumping. Countsobtained were used to calculate the mean conidia loading of seed coatedwith the various treatments. Results obtained using 100% conidia powderwere used as a benchmark and the conidia/carnauba combination powderscompared against it as a determination of efficiency of loading.

Confirmation of conidial viability was achieved by dilution plating onTrichoderma Specific Media (TSM) (see below). A dilution series was setup and duplicate plates inoculated from the series. Colony Forming Units(CFU) counts were made after 7 days, allowing inoculum levels on seedsto be quantified. In addition, fresh, unused conidia were plated toprovide a comparison of before and after seed application.

Germination percentage was also measured. A satisfactory density ofconidia was obtained by spreading approximately 10⁶ conidia in 100 μl onthe media in a 9 cm petri dish. Conidia were incubated in the dark at25° C. for five days, and the area to be observed was then fixed usinglactophenol. Phase contrast microscopy using an inverted compoundmicroscope enabled sufficient examination of the conidia. Conidia wereconsidered viable if germtube lengths were two times the diameter of thepropagule in question. Numbers of germinated and non-germinated conidiain arbitrarily-selected fields of view or in parallel transects, definedwith an ocular micrometer, were counted. A minimum of 300 conidia werecounted to provide an accurate estimate. It is desirable to determinethe viability of propagules on replicate cultures and at variouspositions on the same plate.

This allowed calibration of the seed-coating techniques to obtainsimilar levels of Trichoderma loadings on the seeds for each coatingmethod.

1.3. Seed Germination. One batch (5 seeds) of seeds from each treatmentwas placed on seed test paper (Whatman 181) in a 9 cm Petri dish. Disheswere sealed with Parafilm and held at 20° C. for 7-10 days andgermination rate determined. This was repeated with untreated seed.

Trichoderma Selective Media (adapted from Williams, Clarkson et al 2003)was prepared as follows:

For 1000 ml

Basal Medium Ingredients:

-   -   0.2 g MgSO,    -   0.9 g K₂HPO₄    -   0.15 g KCl    -   1.0 g NH₄NO₃    -   3.0 g glucose    -   0.15 g rose bengal    -   20 g agar    -   950 ml distilled water

Basal Medium Process

Mix liquid ingredients with all solid ingredients, except the agar in a1L Erlenmeyer flask. Add the 20 g agar and stir or shake. Plug withcotton wool and cover with foil. Autoclave.

Biocidal Medium (per litre)

0.25 g crystallized chloramphenicol

0.2 g quintozene

0.2 g captan

1.2 ml propamocarb (Previcur)

50 ml sterile distilled water

Seed Weight

Used as a measure of the homogeneity of the seed batch. Eight replicatesof 25 seeds are weighed and the coefficient of variation (Cv) recorded.This coefficient should not exceed a value of 5. If it does then theprocedure is repeated and the mean of all 16 samples used to calculatethe number of seeds per gram.

Mean TGW Crop Weight (g) SD Cv (g) Cotton 3.138 0.087 2.743 126.46

Results

Direct Enumeration Counts using Haemocytometer

Initial Spore Density of Trichoderma harzianum dry spore preparation (at5% moisture content), determined using haemocytometer, was 7.75×10⁹spores g⁻¹ (n=4, ±2.6×10⁷ 95% CL).

Summary

Cotton seed can be coated with Trichoderma spores in excess the target10⁹ spores seed' for all treatments.

Use of Entostat increases the efficiency of spore delivery as a resultof a reduction in wasted or lost spores.

The germination viability of the spores is unaffected by their use as aseed coating.

Enumeration through direct counting of spores using a haemocytometer orthrough the use of CFU counting gives statistically similar results andtherefore either method may be used once germination viability has beenproved unaffected by the treatment.

As loading is a function of surface area to mass ratio, the loadingpotential of spores on a larger seed may be higher than that shown oncotton.

Effects of seed coating on disease suppression

Seeds are coated with Trichoderma using water or Entostat to achieveloadings of ca. 10⁵ and 10⁶ CFUs seed⁻¹. Water treatments aresuspensions of spores in sterile water in which the seed samples aresoaked for one hour. Seeds are then dried back, a likely commercialscenario, or sown wet coated. Entostat is applied at ratios of 3:1, and9:1, Entostat to spores respectively. Seed treatment methods will thenbe compared on their ability to protect germinating cotton seedlingsfrom Fusarium oxysporum, the causal agent of wilt disease in cotton.

Inoculation of seeds with Trichoderma. Cotton cv. DP69 is inoculated asfollows (target concentration per seed):

-   -   1) Trichoderma at 10⁵/seed using a water suspension (wet        coating)    -   2) Trichoderma at 10⁶/seed using a water suspension (wet        coating)    -   3) Trichoderma at 10⁵/seed using a water suspension (dry        coating)    -   4) Trichoderma at 10⁶/seed using a water suspension (dry        coating)    -   5) Trichoderma at 10⁵/seed using Entostat at 3:1    -   6) Trichoderma at 10⁶/seed using Entostat at 3:1    -   7) Trichoderma at 10⁵/seed using Entostat at 9:1    -   8) Trichoderma at 10⁶/seed using Entostat at 9:1    -   9) No Trichoderma, water only    -   10) No Trichoderma, Entostat only    -   11) Seed only

Enumeration. Trichoderma is quantified using standard dilution platingmethods on Trichoderma specific media. This confirms CFU loadings perseed for treatments 1-8. Dilution platings are carried out in duplicate.

Fusarium bioassay

Inoculum preparation—Fusarium oxysporum is grown on PDA plates fromstock cultures, and incubated at 20° C. to produce actively growingcolonies. Agar plugs are removed from the plates and used to inoculatesterilised (autoclaved at 121° C. for 20 mins) John Innes No.2 pottingmix (80% moisture content; 60 g) mixed with potato cubes (2 mm², 25 g)in 500 ml Erlenmeyer flasks. Flasks are incubated at 20° C. for 14 days.Inoculum levels in the medium are quantified using a dilution platingmethod.

Effectiveness of seed treatment on F. oxysporum. Seeds are sown intoindividual cells of seed trays containing F. oxysporum-inoculated medium(approx. 15 ml/cell). Four replicate batches of ten seeds per treatmentare planted into the cells. Once sown, the trays are placed in a plantgrowth chamber (Weiss Gallenkamp Fitotron SG120) at 20° C. with ca. 16 hlighting. Cells are bottom watered. The number of seedlings survivingare recorded every 3 days for 21 days.

Time to emergence, percentage successful emergence and percentage plantsexpressing symptoms (including lesions and cankers) are recorded and theresults analysed. Differences in Entostat treated seed and untreatedseed are observed.

1-34. (canceled)
 35. A coating composition in powder form for cottonseed, wherein the said coating composition comprises at least oneorganic carrier material in the form of particles having a volume meandiameter of ≧5 μm, wherein the carrier material is selected from waxeshaving a melting point of ≧50° Centigrade, and one or more biologicalagents that possess an activity against at least one pathogen of acotton plant.
 36. The coating composition according to claim 35, whereinthe particles have a volume mean diameter in the range of 10 to 200 μm.37. The coating composition according to claim 35, wherein thebiological agent is selected from a chemical agent and a live biologicalagent or is a mixture thereof.
 38. The coating composition according toclaim 35, wherein the biological agent is selected from chemicalfungicides, arthropodicides and bactericides or is a mixture of two ormore thereof.
 39. The coating composition according to claim 38, whereinthe arthropodicides are insecticides or acaricides.
 40. The coatingcomposition according to claim 35, wherein the organic carrier materialis selected from waxes having a melting temperature ≧60° Centigrade. 41.The coating composition according to claim 35, wherein the organiccarrier material is selected from carnauba wax, beeswax, montan wax,Chinese wax, shellac wax, spermaceti wax, candelilla wax, castor wax,ouricury wax and rice bran wax or is a mixture of two or more thereof.42. A coating composition according to claim 35, wherein the biologicalagent is a live biological agent present in the form of bacterial sporesand/or fungal spores located on the surface of the said particles. 43.Use of an organic carrier, wherein the organic carrier is made up ofparticles of wax having a volume mean diameter ≧5 μm and selected fromnatural waxes, synthetic waxes and mineral waxes having a melting pointof ≧50° Centigrade, in the manufacture of a composition according toclaim
 35. 44. Use according to claim 42, wherein the organic carrier isselected from carnauba wax, beeswax, montan wax, Chinese wax, shellacwax, spermaceti wax, myricyl palmitate, cetyl palmitate, candelilla wax,castor wax, ouricury wax, wool wax, sugar cane wax, retamo wax and ricebran wax or is a mixture of two or more thereof.
 45. Use of wax as anorganic carrier in dry particulate form in a composition for coatingcotton seed according to claim
 35. 46. Use according to claim 45,wherein the organic carrier is selected from natural waxes, syntheticwaxes and mineral waxes having a melting point of ≧50° Centigrade. 47.Use according to claim 44, wherein the organic carrier is selected fromcarnauba wax, beeswax, montan wax, Chinese wax, shellac wax, spermacetiwax, myricyl palmitate, cetyl palmitate, candelilla wax, castor wax,ouricury wax, wool wax, sugar cane wax, retamo wax and rice bran wax oris a mixture of two or more thereof.
 48. A method of manufacturing acomposition for coating cotton seed according to claim 35 thatcomprises: 1) selecting an organic carrier material from waxes having amelting point of ≧50° Centigrade; 2) comminuting said organic carriermaterial into particles of a volume mean diameter ≧5 μm; and 3) addingto the particles a biological agent that possesses an activity againstat least one pathogen of a cotton plant.
 49. The method according toclaim 48, wherein the organic carrier material is selected from carnaubawax, beeswax, montan wax, Chinese wax, shellac wax, spermaceti wax,myricyl palmitate, cetyl palmitate, candelilla wax, castor wax, ouricurywax, wool wax, sugar cane wax, retamo wax and rice bran wax or is amixture of two or more thereof.
 50. The method according to claim 48,wherein the organic carrier material is carnauba wax.
 51. A cotton seedcomprising a coating composition according to claim
 35. 52. A method ofcoating cotton seed with a coating composition that comprises an organiccarrier material in the form of an organic wax having a melting point of≧50° Centigrade, wherein the organic wax is in dry particulate form andthe wax particles have a volume mean diameter of ≧5 μm, and a biologicalagent that has an activity against a cotton plant pathogen selected froma fungal pathogen, a bacterial pathogen and an arthropod pathogen, themethod comprising adding the biological agent to the organic carriermaterial, mixing the two together and applying the resulting compositionto cotton seeds.
 53. The method according to claim 52, wherein the waxis selected from carnauba wax, beeswax, montan wax, Chinese wax, shellacwax, spermaceti wax, candelilla wax, castor wax, ouricury wax and ricebran wax or is a mixture of two or more thereof.
 54. The methodaccording to claim 52, wherein biological agent is selected fromchemical insecticides and acaricides, fungicides, bactericides and livebiological agents.